Method of treating inflammation, allergy and asthma with a purified extract (ATC2) isolated from Pseudolysimachion rotundam var. subintegrum containing abundant amount of active ingredient

ABSTRACT

The present invention relates to inventive novel industrialized method for preparing purified extract containing more abundant active ingredients such as catalpol derivatives from the extract of  Pseudolysimachion rotundum  var  subintegrum  than that prepared by the conventional preparation method disclosed in the prior art and the therapeutics or functional health food comprising the purified extract for treating and preventing inflammatory, allergic or asthmatic disease. The purified extract showed more potent anti-inflammatory, anti-allergy and anti-asthma activity than that prepared by the conventional preparation method disclosed in the prior art through various in vivo tests such as inhibition test on the reproduction of eosinophil, the release of immunoglobulin and inflammatory chemokines in plasma and bronchoalveolar fluid as well as the suppression of airway hyperresponsiveness and goblet cell hyperplasia in a OVA-sensitized/challenged mouse model.

This application is a Division of and claims priority upon U.S.application Ser. No. 14/653,852 filed on Jun. 19, 2015 (pending), whichis a U.S. National Phase Patent Application under 35 U.S.C. §371 ofInternational Application PCT/KR2013/011986, filed on Dec. 23, 2013,which claims priority to Korean Patent Application No. 10-2013-0084167,filed on Jul. 17, 2013 and Korean Patent Application No.10-2012-0158130, filed on Dec. 31, 2012. All publications, patents,patent applications, databases and other references cited in thisapplication, all related applications referenced herein, and allreferences cited therein, are incorporated by reference in theirentirety as if restated here in full and as if each individualpublication, patent, patent application, database or other referencewere specifically and individually indicated to be incorporated byreference.

TECHNICAL FIELD

The present invention relates to a purified extract isolated fromPseudolysimachion rotundum var. subintegrum containing abundant amountof active ingredient, the preparation thereof, and the compositioncomprising the same as an active ingredient for preventing or treatinginflammation, allergy and asthma.

BACKGROUND ART

Generally, an inflammatory response is a normal response of human bodyassociated with an edema, a pain etc in case that a tissue or a cellreceived any invasion causing some organic change in the tissue or cell.Recently, various kinds of cytokines have been found to be involved inthe inflammatory disease.

Allergic reaction may be classified into four categories, i.e., type I,II, III and IV according to the types of response or two categories,i.e., immediate type allergic reaction such as type I, II or III, anddelayed type allergic reaction such as type IV according to the types ofthe period from the re-sensitization time caused by allergen to theonset time of reaction.

Among them, type I allergy, being involved in IgE antibody and called asanaphylaxis type allergy, causes to a bronchial asthma, atopic diseasessuch as dermatitis or gastroenteritis etc, allergic rhinitis such aspollenosis, allergic conjunctivitis, food allergy and the like.

Asthma is regarded as a complex syndrome of the airways that ischaracterized by various clinical symptoms, for example, cough, dyspneacaused by airflow obstruction, acute or chronic airway inflammation,airway hyperresponsiveness (AHR) and structural remodeling and can bereversibly or irreversibly recoverable. Most of asthma is allergicdisease and is characterized by chronic airway inflammation andbronchial hyperresponsiveness (Minoguchi K and Adachi M.,Pathophysiology of asthma. In: Cherniack N S, Altose M D, Homma I.editors. Rehabilitation of the patient with respiratory disease. NewYork: McGraw-Hill, 1999, pp 97-104).

The asthma can be classified two types, i.e., extrinsic asthma andintrinsic asthma. The extrinsic asthma is caused by exposing antigen andit is shown positive reaction in skin test or bronchial provocation testagainst the antigen. Usually causing ages is getting younger. It ismainly caused by House Dust Mite Dermatophagoides and pollen, epitheliumof animal, fungi and so on. The intrinsic asthma is caused by upperrespiratory infections, exercise, emotional instability, changing ofclimate of humidity and it is common to adult patient. Also, the IgEantigen of extrinsic asthma can be detected by skin test due toincreasing IgE in serum.

With regards to pathophysiology, asthma is recognized by T-helper2(Th2)-cell-driven chronic inflammation, and a variety of inflammatorymediators, such as cytokines, chemokines, signaling molecules, adhesionmolecules and growth factors, from immune cells and structural cells inthe airways are involved in various stages of asthma (Elias J A et al.,J Clin Invest., 111, pp 291-7, 2003). The activated inflammatory cellssuch as eosinophil, mast cells, alveolar macrophage etc in the bronchusof patients suffering from asthma, release various inflammatorymediators such as cystein leukotrienes, prostaglandins etc and isinvolved in potent bronchial constriction (Maggi E., Immunotechnology 3,pp 233-244, 1998; Pawankar R. Curr. Opin. Allergy Clin. Immunol., 1, pp3-6, 2001; Barnes P J et al., Phamacol. Rev. 50, pp 515-596, 1998).

Accordingly, since the reproduction of various cytokines involved ininflammatory cell activation, such as IL-4, IL-5, IL-13 etc and IgE andreproduction of cystein leukotrienes released from the inflammatorycells are the main causes of inflammation, allergic reaction and asthma,there have been much studied to develop the inhibiting agents from thereproduction of those till now.

The present inventors have been focused to develop potent treating agentderived from natural resources with safety and efficacy such as plant,animals etc having potent inhibiting activity from the reproduction ofinflammatory cells and finally, have found that the extract ofPseudolysimachion longifolium showed potent anti-inflammatory,anti-allergy and anti-asthma activity (Korean Patent No. 10-860080) andvarious compounds isolated therefrom such as, verproside(6-O-3,4-dihydroxybenzoyl catalpol), picroside II(6-O-4-hydroxy-3-methoxybenzoyl catalpol), verminoside(6-O-3,4-Dihydroxy cinnamoyl catalpol), 6-O-veratroyl catalpol(6-O-3,4-Dimethoxy benzoyl catalpol), minecoside(6-O-3-hydroxy-4-methoxycinnamoyl catalpol), catalpol and the like, alsoshowed potent anti-inflammatory, anti-allergy and anti-asthma activity(Korean Patent Publication No. 10-2006-125499).

However, there have been difficulties in mass-production andindustrialization using by the extract of Pseudolysimachion longifoliumsince the plant extract contains very little active ingredients such ascatalopol derivatives.

Pseudolysimachion rotundum var subintegrum, is a perennial herbdistributed in Korea, China, Japan, Ostrov Sakhalin, and Russia.

Based on the previous studies on the anti-inflammatory, anti-allergy andanti-asthma activity of the extract of Pseudolysimachion longifoliumdisclosed in Korean Patent No. 10-860080, the present inventors havetried to develop more efficient method for preparing more potent andmore abundant ingredients showing anti-inflammatory, anti-allergy andanti-asthma activity isolated from the extract of Pseudolysimachionrotundum var subintegrum.

However, there has been not reported or disclosed about the efficientmethod for preparing more potent and more abundant ingredients showinganti-inflammatory, anti-allergy and anti-asthma activity isolated fromthe extract of Pseudolysimachion rotundum var subintegrum than those inthe above cited literatures, the disclosures of which are incorporatedherein by reference.

Accordingly, the present inventors have found the novel industrializedmethod for preparing purified extract containing more abundant activeingredients such as catalpol derivatives from the extract ofPseudolysimachion rotundum var subintegrum and the purified extractshowed more potent anti-inflammatory, anti-allergy and anti-asthmaactivity than that prepared by the conventional preparation methoddisclosed in the prior art through various in vivo tests such asinhibition test on the reproduction of eosinolphil, the release ofimmunoglobulin and inflammatory chemokines in plasma and bronchoalveolarfluid as well as the suppression of airway hyperresponsiveness andgolblet cell hyperplasia in a OVA-sensitized/challenged mouse model.

DISCLOSURE Technical Problem

The present invention provides a novel method for preparing purifiedextract containing abundant active ingredients such as catalpolderivatives from Pseudolysimachion rotundum var subintegrum and theextract prepared thereby.

The present invention also provides a pharmaceutical composition and ahealth food comprising the purified extract containing abundant activeingredients such as catalpol derivatives from Pseudolysimachion rotundumvar subintegrum as an active ingredient in an effective amount to treatand prevent inflammatory, allergic or asthmatic disease.

The present invention also provides a use of a purified extractcontaining abundant active ingredients such as catalpol derivatives fromPseudolysimachion rotundum var subintegrum showing anti-inflammatory,anti-allergic and anti-asthmatic activity.

The present invention also provides a method of treating or preventinginflammatory, allergic or asthmatic disease in a mammal comprisingadministering to said mammal an effective amount of purified extractcontaining abundant active ingredients such as catalpol derivatives fromPseudolysimachion rotundum var subintegrum, together with apharmaceutically acceptable carrier thereof.

Technical Solution

Accordingly, it is an object of the present invention to provide novelpurified extract containing abundant catalpol derivatives from theextract of Pseudolysimachion rotundum var subintegrum.

The term “catalpol derivatives” disclosed herein comprises verproside,catalposide, picroside II, isovanilloyl catalpol and 6-O-veratroylcatalpol etc.

The term “Pseudolysimachion rotundum var subintegrum” disclosed hereincomprises the cultivated or naturally grown plant and commerciallyavailable plant, but not intent to limit thereto herein.

The term “novel purified extract” disclosed herein comprises (a) thepurified extract fractionated with butanol (designated as “ATC1”hereinafter) and (b) the purified extract with the secondaryfractionation (designated as “ATC2” hereinafter).

Specifically, the term “the purified extract fractionated with butanol(ATC1)” is characterized by containing 15-50% (w/w) verproside, 0.3-10%(w/w) veratric acid, 0.5-10% (w/w) catalposide, 0.3-10% (w/w) picrosideII, 0.3-10% (w/w) isovanilloyl catalpol and 0.3-10% (w/w) 6-O-veratroylcatalpol based on the weight of total extract (100%) ofPseudolysimachion rotundum var subintegrum; preferably, 20-25% (w/w)verproside, 0.5-5% (w/w) veratric acid, 1-5% (w/w) catalposide, 0.5-5%(w/w) picroside II, 0.5-5% (w/w) isovanilloyl catalpol and 1-5% (w/w)6-O-veratroyl catalpol based on the weight of total extract (100%) ofPseudolysimachion rotundum var subintegrum; or characterized bycontaining 12.3-47% (w/w) catalposide derivatives in total extract(100%) of Pseudolysimachion rotundum var subintegrum and having therelative mixed ratio (w/w) between the weight of each catalposidederivative, of 15.0-18.0 (w/w) verproside, 2.10-2.60 (w/w) catalposide,1 (w/w) picroside II, 1.00-1.30 (w/w) isovanilloyl catalpol and2.00-2.30 (w/w) 6-O-veratroyl catalpol; preferably, 16.0-17.0 (w/w)verproside, 2.20-2.50 (w/w) catalposide, 1 (w/w) picroside II, 1.10-1.20(w/w) isovanilloyl catalpol and 2.10-2.20 (w/w) 6-O-veratroyl catalpol;more preferably, 16.20-16.99 (w/w) verproside, 2.40-2.45 (w/w)catalposide, 1 (w/w) picroside II, 1.10-1.19 (w/w) isovanilloyl catalpoland 2.10-2.19 (w/w) 6-O-veratroyl catalpol.

More specifically, the term “the purified extract fractionated withbutanol (ATC1)” is characterized by being prepared by the process of;adding at least one extracting solvent selected from water, C1-C4 loweralcohol such as methanol, ethanol, butanol etc or the mixtures thereof,preferably, mixture of water and ethanol, more preferably, 30-80 (w/w)ethanol in water to dried Pseudolysimachion rotundum var subintegrum atthe 1st step; subjecting to at least one extraction method selected fromreflux extraction with hot water, cold water extraction,ultra-sonication or conventional extraction, preferably cold waterextraction followed by reflux extraction at the temperature ranging from10 to 100° C., preferably from 20 to 90° C., for the period ranging from30 mins to 72 hours, preferably, 6 to 48 hours, more preferably, coldwater extraction at the temperature ranging from 10 to 60° C.,preferably from 20 to 50° C., for the period ranging from 30 mins to 72hours, preferably, 6 to 48 hours and then reflux extraction at thetemperature ranging from 40 to 120° C., preferably from 60 to 90° C.,for the period ranging from 30 mins to 72 hours, preferably, 6 to 48hours, repeatedly, to afford the 1st extract at 2nd step; suspending the1st extract in about 0.5-10 fold volume (v/v), preferably, about 1-5fold volume (v/v) of water to afford suspended extract at 3rd step; andadding about 0.5-20 fold volume (v/v), preferably, about 1-10 foldvolume (v/v) of butanol, fractionating into water layer and butanollayer and collecting the butanol layer to afford the purified extractfractionated with butanol (ATC1) containing 15-50% (w/w) verproside,0.3-10% (w/w) veratric acid, 0.5-10% (w/w) catalposide, 0.3-10% (w/w)picroside II, 0.3-10% (w/w) isovanilloyl catalpol and 0.3-10% (w/w)6-O-veratroyl catalpol based on the weight of total extract (100%) ofPseudolysimachion rotundum var subintegrum to treat and preventinflammatory, allergic or asthmatic disease.

Accordingly, in an another embodiment of the present invention, thepresent invention also provides a method for preparing the purifiedextract fractionated with butanol (ATC1) isolated from Pseudolysimachionrotundum var subintegrum comprising the steps of; adding at least oneextracting solvent selected from water, C1-C4 lower alcohol such asmethanol, ethanol, butanol etc or the mixtures thereof, preferably,mixture of water and ethanol, more preferably, 30-80 (w/w) ethanol inwater to dried Pseudolysimachion rotundum var subintegrum at the 1ststep; subjecting to at least one extraction method selected from refluxextraction with hot water, cold water extraction, ultra-sonication orconventional extraction, preferably cold water extraction followed byreflux extraction at the temperature ranging from 10 to 100° C.,preferably from 20 to 90° C., for the period ranging from 30 mins to 72hours, preferably, 6 to 48 hours, more preferably, cold water extractionat the temperature ranging from 10 to 60° C., preferably from 20 to 50°C., for the period ranging from 30 mins to 72 hours, preferably, 6 to 48hours and then reflux extraction at the temperature ranging from 40 to120° C., preferably from 60 to 90° C., for the period ranging from 30mins to 72 hours, preferably, 6 to 48 hours, repeatedly, to afford the1st extract at 2nd step; suspending the 1st extract in about 0.5-10 foldvolume (v/v), preferably, about 1-5 fold volume (v/v) of water to affordsuspended extract at 3rd step; and adding about 0.5-20 fold volume(v/v), preferably, about 1-10 fold volume (v/v) of butanol,fractionating into water layer and butanol layer and collecting thebutanol layer to afford the purified extract fractionated with butanol(ATC1) containing 15-50% (w/w) verproside, 0.3-10% (w/w) veratric acid,0.5-10% (w/w) catalposide, 0.3-10% (w/w) picroside II, 0.3-10% (w/w)isovanilloyl catalpol and 0.3-10% (w/w) 6-O-veratroyl catalpol based onthe weight of total extract (100%) of Pseudolysimachion rotundum varsubintegrum to treat and prevent inflammatory, allergic or asthmaticdisease.

Specifically, the term “the purified extract with the secondaryfractionation (ATC2)” is characterized by containing 30-60% (w/w)verproside, 0.5-10% (w/w) veratric acid, 2-20% (w/w) catalposide, 1-10%(w/w) picroside II, 1-10% (w/w) isovanilloyl catalpol and 2-20% (w/w)6-O-veratroyl catalpol based on the weight of total extract (100%) ofPseudolysimachion rotundum var subintegrum; preferably, 40-50% (w/w)verproside, 1-5% (w/w) veratric acid, 3-10% (w/w) catalposide, 2-5%(w/w) picroside II, 2-8% (w/w) isovanilloyl catalpol and 3-8% (w/w)6-O-veratroyl catalpol based on the weight of total extract (100%) ofPseudolysimachion rotundum var subintegrum; or characterized bycontaining 36.5-91% (w/w) catalposide derivatives in total extract(100%) of Pseudolysimachion rotundum var subintegrum and having therelative mixed ratio (w/w) between the weight of each catalposidederivative, of 13.0-16.0 (w/w) verproside, 2.20-2.50 (w/w) catalposide,1 (w/w) picroside II, 1.10-1.40 (w/w) isovanilloyl catalpol and2.00-2.20 (w/w) 6-O-veratroyl catalpol; preferably, 14.0-15.0 (w/w)verproside, 2.30-2.45 (w/w) catalposide, 1 (w/w) picroside II, 1.20-1.35(w/w) isovanilloyl catalpol and 2.00-2.10 (w/w) 6-O-veratroyl catalpol;more preferably, 14.50-14.99 (w/w) verproside, 2.35-2.43 (w/w)catalposide, 1 (w/w) picroside II, 1.25-1.34 (w/w) isovanilloyl catalpoland 2.01-2.09 (w/w) 6-O-veratroyl catalpol.

More specifically, the term “the purified extract with the secondaryfractionation (ATC2)” is characterized by being prepared by the processof adding at least one extracting solvent selected from water, C1-C4lower alcohol such as methanol, ethanol, butanol etc or the mixturesthereof, preferably, mixture of water and ethanol, more preferably,30-80 (w/w) ethanol in water to dried Pseudolysimachion rotundum varsubintegrum at the 1st step; subjecting to at least one extractionmethod selected from reflux extraction with hot water, cold waterextraction, ultra-sonication or conventional extraction, preferably coldwater extraction followed by reflux extraction at the temperatureranging from 10 to 100° C., preferably from 20 to 90° C., for the periodranging from 30 mins to 72 hours, preferably, 6 to 48 hours, morepreferably, cold water extraction at the temperature ranging from 10 to60° C., preferably from 20 to 50° C., for the period ranging from 30mins to 72 hours, preferably, 6 to 48 hours and then reflux extractionat the temperature ranging from 40 to 120° C., preferably from 60 to 90°C., for the period ranging from 30 mins to 72 hours, preferably, 6 to 48hours, repeatedly, to afford the 1st extract at 2nd step; suspending the1st extract in about 0.5-10 fold volume (v/v), preferably, about 1-5fold volume (v/v) of water to afford suspended extract at 3rd step;adding about 0.5-20 fold volume (v/v), preferably, about 1-10 foldvolume (v/v) of butanol, fractionating into water layer and butanollayer and collecting the butanol layer to afford the purified extractfractionated with butanol (ATC1) at the 3rd step; and subjecting thepurified extract fractionated with butanol (ATC1) to at least onepurification process selected from the group consisting of reverse phasepartition chromatography, normal phase partition chromatography, ionexchange chromatography, and size exclusion chromatography to afford thepurified extract with the secondary fractionation (ATC2) containing30-60% (w/w) verproside, 0.5-10% (w/w) veratric acid, 2-20% (w/w)catalposide, 1-10% (w/w) picroside II, 1-10% (w/w) isovanilloyl catalpoland 2-20% (w/w) 6-O-veratroyl catalpol based on the weight of totalextract (100%) of Pseudolysimachion rotundum var subintegrum to treatand prevent inflammatory, allergic or asthmatic disease.

Accordingly, in an another embodiment of the present invention, thepresent invention also provides a method for preparing the purifiedextract with the secondary fractionation (ATC2) isolated fromPseudolysimachion rotundum var subintegrum comprising the steps of;adding at least one extracting solvent selected from water, C1-C4 loweralcohol such as methanol, ethanol, butanol etc or the mixtures thereof,preferably, mixture of water and ethanol, more preferably, 30-80 (w/w)ethanol in water to dried Pseudolysimachion rotundum var subintegrum atthe 1st step; subjecting to at least one extraction method selected fromreflux extraction with hot water, cold water extraction,ultra-sonication or conventional extraction, preferably cold waterextraction followed by reflux extraction at the temperature ranging from10 to 100° C., preferably from 20 to 90° C., for the period ranging from30 mins to 72 hours, preferably, 6 to 48 hours, more preferably, coldwater extraction at the temperature ranging from 10 to 60° C.,preferably from 20 to 50° C., for the period ranging from 30 mins to 72hours, preferably, 6 to 48 hours and then reflux extraction at thetemperature ranging from 40 to 120° C., preferably from 60 to 90° C.,for the period ranging from 30 mins to 72 hours, preferably, 6 to 48hours, repeatedly, to afford the 1st extract at 2nd step; suspending the1st extract in about 0.5-10 fold volume (v/v), preferably, about 1-5fold volume (v/v) of water to afford suspended extract at 3rd step;adding about 0.5-20 fold volume (v/v), preferably, about 1-10 foldvolume (v/v) of butanol, fractionating into water layer and butanollayer and collecting the butanol layer to afford the purified extractfractionated with butanol (ATC1) at the 3rd step; and subjecting thepurified extract fractionated with butanol (ATC1) to at least onefurther purification process selected from the group consisting ofreverse phase partition chromatography, normal phase partitionchromatography, ion exchange chromatography, and size exclusionchromatography to afford the purified extract with the secondaryfractionation (ATC2) containing 30-60% (w/w) verproside, 0.5-10% (w/w)veratric acid, 2-20% (w/w) catalposide, 1-10% (w/w) picroside II, 1-10%(w/w) isovanilloyl catalpol and 2-20% (w/w) 6-O-veratroyl catalpol basedon the weight of total extract (100%) of Pseudolysimachion rotundum varsubintegrum to treat and prevent inflammatory, allergic or asthmaticdisease.

Specifically, the term “further purification process” is selected from(i) reverse phase partition chromatography, (ii) normal phase partitionchromatography, (iii) ion exchange chromatography or (iv) size exclusionchromatography, preferably, reverse phase partition chromatography orany chromatography using by any resin as a stationary phase which canretain non-polar substance while eluting polar substance, for example,Sephadex resin such as Sephadex, Sephadex LH20, Sephadex G-25, SephadexG-10, Sepharose, Superdex, methylacrylate resin, carboxymethylcellulose, sulphopropyl cellulose, carboxymethyl Sephadex, sulphopropylSephadex, carboxymethyl Sepharose, sulphopropyl Sepharose and the like;reverse polymer resin using by Stylene-divinylbenzen co-polymer such asPolymer X, HP20, PRP-h1 Polymer and the like or Methacrylate supportresin etc; normal Silica gel such as BPC (Bonded phase chromatography)product, Silica product procured from YMC Co. Ltd, Silica productprocured from DAISO Co. Ltd, Silica product procured from ASAHI Co. Ltd,Silica product procured from COSMOSYL Co. Ltd and the like; ODS productused for HPLC filler such as ODS product procured from YMC Co. Ltd, ODSproduct procured from DAISO Co. Ltd, ODS product procured from ASAHI Co.Ltd, ODS product procured from CHEMCO Co. Ltd, ODS product procured fromMerck Co. Ltd ODS product procured from COSMOSYL Co. Ltd ODS productprocured from FUJI Co. Ltd and the like.

In a preferred embodiment adopting (i) reverse phase partitionchromatography as a further purification process of the presentinvention, the “stationary phase in the above-described reverse phasepartition chromatography” may be any stationary phases such as reversephase substance as a stationary phase which can retain non-polarsubstance while eluting polar substance, preferably, Silica gel basedstationary phase, polymer based stationary phase such as polystyrene etcand the like, more preferably, Silica gel derivatives such as C2, C4,C6, C8, C10, C12, 14, C16, C18 and the like; or a polymer basedstationary phase such as PS-2, Oasis HLB and the like, more and morepreferably, reverse phase Silica gel (C18(IV)-D), ODS-A/ODS-AQ productfrom YMC Co. Ltd., SP-C-ODS product from CHEMCO Co. Ltd., SP-ODS-RPSproduct from DAISO Co. Ltd., 5C18 product from COSMOSYL Co. Ltd.,Chromatorex product from FUJI Co. Ltd., etc.

In a preferred embodiment adopting (i) reverse phase partitionchromatography as a further purification process of the presentinvention, the “mobile phase in the above-described (i) reverse phasepartition chromatography” may be at least one solvent selected fromwater, acetonitrile, lower alcohol such as methanol, ethanol, butanoletc, tetrahydrofuran (THF) or the mixture thereof, preferably, water,lower alcohol such as methanol, ethanol, butanol etc, or the mixturethereof, more preferably, the mixture solvent of water and methanol,more and more preferably, the mixture solvent of water and methanol withstarting from 90:10 (v/v) to 60:40 (v/v) to elute polar substance.

In a preferred embodiment adopting (ii) normal phase partitionchromatography as a further purification process of the presentinvention, the “stationary phase in the above-described normal phasepartition chromatography” may be any stationary phases such as normalphase substance as a stationary phase which can retain polar substancewhile eluting non-polar substance, preferably, Silica gel, Fluorosyl, oralumina based stationary phase, CN, Diol, or NH2 moiey polymer basedstationary phase and the like, more preferably, Silica gel, Fluorosyl,or alumina based stationary phase, etc.

In a preferred embodiment adopting (ii) normal phase partitionchromatography as a further purification process of the presentinvention, the “mobile phase in the above-described (ii) normal phasepartition chromatography” may be at least one solvent selected fromhexane, heptane, ethylacetate, ethanol, diethylether, 2-propanol or themixture thereof, preferably, hexane, heptane, ethylacetate or themixture thereof to elute non-polar substance.

In a preferred embodiment adopting (iii) ion exchange chromatography asa further purification process of the present invention, the “stationaryphase in the above-described (iii) ion exchange chromatography” may beany high molecular stationary phases as a stationary phase which havecharged cross-linking moiety, preferably, cation exchange resin, anionexchange resin, or synthetic adsorbent, and the like, more preferably,strongly acidic cation exchange resin such as AG 50W-x8, AmberliteIR-120, Dowex 60W-x8, SKIB etc; weakly acidic cation exchange resin suchas Amberlite IRA-67, Dowex 3-x4A etc; strongly basic cation exchangeresin such as DIAION SKIB, DIAION PK216, DIAION CR20, DIAION UBK555(Mitsubishi Chemical Co.), TRILITE SPC 160H, TRILITE SPC 180H, TRILITESPC 400JH (Samyang Co. Ltd.), AMBERLITE 200C Na, AMBERLITE CG50,AMBERLITE CR1310 Na, AMBERJET 200H, AMBERLYST 131 WET, ALBERLYST 232 WET(ROHM and HAAS Co. Ltd.), Lewatit VP OC 1800, Lewatit VP OC 1812,Lewatit MD51368 Na, Lewaitit K1221 (Bayer Co. Ltd.), PUROLITE PCR833CA,PUROLITE C145 (Purolite Co. Ltd.), MFG210, MFG 250 (Finex Co. Ltd.) etc;strongly basic anion exchange resin such as SA11A, SA20A, SA21A etc; orCaptoQ (GE Healthcare Co. Ltd.), or the resin having similar propertythereto such as Toyopearl QEA (Tosoh Co. Ltd.), Q Sepharose FF (GEHealthcare Co. Ltd.), Fractogel EMD, Fractogel TMAE, Fractogel HICAP(Merck KGaA Co. Ltd or Darmstadt Co. Ltd.); more and more preferably,SA21A; adsorbent such as SP207, HP20SS, HP20 etc, more preferably, HP20.

In a preferred embodiment adopting (iv) size exclusion chromatography asa further purification process of the present invention, the “stationaryphase in the above-described (iv) size exclusion chromatography” may beany gel type stationary phases as a stationary phase which can separateby the size of sample, preferably, dextran-based gel such as sephadex(for example, sephadex G-25), polyacrylamide-based gel such as Sephacryl(for example, Sephacryl-S400), Agarose-based gel such as Superose orSepharose (for example, Sepharose CL-4B) or the combinations thereofsuch as Superdex 200 combination Dextran (For example, Sephadex™), orcross-linked Agarose gel (Superose™) and the like, however it shall benot limited thereto herein. The “mobile phase in the above-described(iv) size exclusion chromatography” may be buffer solution selected fromthe group consisting of sodium acetate buffer, sodium phosphate buffer,ammonium acetate buffer, MES (2-(N-morpholino)ethanesulphonic acid),Bis-Tris[2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propandiol],ADA [N-(2-acetamido)iminodiacetate), PIPES[piperaxine-N,N′-Bis(2-ethanesulophonic acid)], BES[N.N′-Bis(2-hydroxyethyl)-2-aminoethansulphonic acid), MOPS[3-(N-morpholino)propansulphonic acid)], TES(N-Tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid], HEPES[N-2-hydroxyethyl-piperazine-N′-2-ethanesulphonic acid), and the like;preferably, sodium acetate buffer, sodium phosphate buffer, or ammoniumacetate buffer.

In a preferred embodiment of the present invention, the presentinvention can also perform (v) Gel permeation chromatography or Gelfiltration chromatography in addition to (i) reverse phase partitionchromatography, (ii) normal phase partition chromatography, (iii) ionexchange chromatography, (iv) size exclusion chromatography or thecombination thereof, as a further purification process disclosed herein.

The present invention also provides novel purified extract such as (a)the purified extract fractionated with butanol (designated as “ATC1”hereinafter) or (b) the purified extract with the secondaryfractionation (designated as “ATC2” hereinafter) prepared by theabove-described preparation methods.

The present invention also provides novel purified extract fractionatedwith butanol (ATC1) from the extract of Pseudolysimachion rotundum varsubintegrum, prepared by the above-described preparation methods, whichcontains 12.3-47% (w/w) catalposide derivatives in total extract (100%)of Pseudolysimachion rotundum var subintegrum wherein said catalposidederivatives consist of 15-50% (w/w) verproside, 0.3-10% (w/w) veratricacid, 0.5-10% (w/w) catalposide, 0.3-10% (w/w) picroside II, 0.3-10%(w/w) isovanilloyl catalpol and 0.3-10% (w/w) 6-O-veratroyl catalpol,preferably, 20-25% (w/w) verproside, 0.5-5% (w/w) veratric acid, 1-5%(w/w) catalposide, 0.5-5% (w/w) picroside II, 0.5-5% (w/w) isovanilloylcatalpol and 1-5% (w/w) 6-O-veratroyl catalpol based on the weight oftotal extract (100%) of Pseudolysimachion rotundum var subintegrum.

The present invention also provides novel purified extract fractionatedwith butanol (ATC1) from the extract of Pseudolysimachion rotundum varsubintegrum, prepared by the above-described preparation methods, whichshows the relative mixed ratio (w/w) between the weight of eachcatalposide derivative of 15.0-18.0 (w/w) verproside, 2.10-2.60 (w/w)catalposide, 1 (w/w) picroside II, 1.00-1.30 (w/w) isovanilloyl catalpoland 2.00-2.30 (w/w) 6-O-veratroyl catalpol; preferably, 16.0-17.0 (w/w)verproside, 2.20-2.50 (w/w) catalposide, 1 (w/w) picroside II, 1.10-1.20(w/w) isovanilloyl catalpol and 2.10-2.20 (w/w) 6-O-veratroyl catalpol;more preferably, 16.20-16.99 (w/w) verproside, 2.40-2.45 (w/w)catalposide, 1 (w/w) picroside II, 1.10-1.19 (w/w) isovanilloyl catalpoland 2.10-2.19 (w/w) 6-O-veratroyl catalpol.

The present invention also provides novel purified extract with thesecondary fractionation (ATC2) from the extract of Pseudolysimachionrotundum var subintegrum, prepared by the above-described preparationmethods, which contains 36.5-91% (w/w) catalposide derivatives in totalextract (100%) of Pseudolysimachion rotundum var subintegrum, whereinsaid catalposide derivatives consist of 30-60% (w/w) verproside, 0.5-10%(w/w) veratric acid, 2-20% (w/w) catalposide, 1-10% (w/w) picroside II,1-10% (w/w) isovanilloyl catalpol and 2-20% (w/w) 6-O-veratroyl catalpolbased on the weight of total extract (100%) of Pseudolysimachionrotundum var subintegrum; preferably, 40-50% (w/w) verproside, 1-5%(w/w) veratric acid, 3-10% (w/w) catalposide, 2-5% (w/w) picroside II,2-8% (w/w) isovanilloyl catalpol and 3-8% (w/w) 6-O-veratroyl catalpolbased on the weight of total extract (100%) of Pseudolysimachionrotundum var subintegrum.

The present invention also provides novel purified extract with thesecondary fractionation (ATC2) from the extract of Pseudolysimachionrotundum var subintegrum, prepared by the above-described preparationmethods, which show the relative mixed ratio (w/w) between the weight ofeach catalposide derivative, of 13.0-16.0 (w/w) verproside, 2.20-2.50(w/w) catalposide, 1 (w/w) picroside II, 1.10-1.40 (w/w) isovanilloylcatalpol and 2.00-2.20 (w/w) 6-O-veratroyl catalpol; preferably,14.0-15.0 (w/w) verproside, 2.30-2.45 (w/w) catalposide, 1 (w/w)picroside II, 1.20-1.35 (w/w) isovanilloyl catalpol and 2.00-2.10 (w/w)6-O-veratroyl catalpol; more preferably, 14.50-14.99 (w/w) verproside,2.35-2.43 (w/w) catalposide, 1 (w/w) picroside II, 1.15-1.24 (w/w)isovanilloyl catalpol and 2.01-2.09 (w/w) 6-O-veratroyl catalpol.

The term “purified extract” disclosed herein may be used as a dried formprepared by the vacuum evaporation method, freeze dry method or hot-airdrying method etc.

The term “inflammatory disease” disclosed herein comprises eczema,atopic dermatitis, conjunctivitis, periodontal disease, rhinitis, otitismedia, laryngopharyngitis, tonsillitis, pneumonia, gastric ulcer,gastritis, Crohn's disease, colitis, hemorrhoid, gout, ankylosingspondylitis, rheumatic fever, systemic lupus erythematosus,fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatic arthritis,periarthritis of shoulder, tendinitis, tenosynovitis, peritendinitis,myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiplesclerosis, chronic inflammatory disease, acute inflammatory disease etc,but not intended herein to limit thereto, preferably, eczema, atopicdermatitis, rheumatic arthritis, chronic inflammatory disease, acuteinflammatory disease etc,

The term “allergic disease” disclosed herein comprises allergicrhinitis, allergic dermatitis, contact dermatitis, hives, insectallergy, food allergy, drug allergy, allergic conjunctivitis,hypersensitivity etc, but not intended herein to limit thereto,preferably, allergic rhinitis, allergic dermatitis, contact dermatitis,hives, insect allergy, food allergy, drug allergy, more preferably,allergic dermatitis, contact dermatitis.

The term “asthmatic disease” disclosed herein comprises any asthmacaused by various external factors, but not intended herein to limitthereto, such as dust mites, animal's fur or dandruff, cockroaches,food, drug, cough, cigarette smoke, air pollution, food additive,physical activity such as exercise etc, weather change, yellow sand,stress etc.

The term “prevent” disclosed herein comprises any act to inhibit orpostpone the occurrence of certain disease or disorder disclosed hereinby way of administrating the inventive composition; and the term “treat”disclosed herein comprises any act to alleviate or favorably changingthe symptom associated with certain disease or disorder disclosed hereinby way of administrating the inventive composition.

The present inventors have found that the novel industrialized methodfor preparing purified extract can provide more abundant activeingredients, i.e., 36.5% to 91.0% (w/w) such as catalpol derivativesfrom the extract of Pseudolysimachion rotundum var subintegrum comparingwith the crude extract prepared by the conventional method disclosed inthe prior art wherein the content of catalpol derivatives in only 8.49%(w/w) through various HPLC analyses, for example, the inventive purifiedextract (ATC1) contains 17.60% (w/w) verproside, 0.72% (w/w) veratricacid, 2.62% (w/w) catalposide, 1.08% (w/w) picroside II, 1.26% (w/w)isovanilloyl catalpol and 2.36% (w/w) 6-O-veratroyl catalpol (SeeExample 2) and the inventive purified extract (ATC2) contains 43.83%(w/w) verproside, 1.80% (w/w) veratric acid, 7.07% (w/w) catalposide,2.93% (w/w) picroside II, 3.85% (w/w) isovanilloyl catalpol and 6.15%(w/w) 6-O-veratroyl catalpol while the crude extract (CX) prepared bythe conventional method disclosed in the prior art contains only 5.9%(w/w) verproside, 0.21% (w/w) veratric acid, 0.82% (w/w) catalposide,0.40% (w/w) picroside II, 0.42% (w/w) isovanilloyl catalpol and 0.74%(w/w) 6-O-veratroyl catalpol based on the weight of total extract (100%)of Pseudolysimachion rotundum var subintegrum; crude extract; as well asthe inventive purified extract showed more potent anti-inflammatory,anti-allergy and anti-asthma activity than that prepared by theconventional preparation method through various in vivo test such asinhibition test on the reproduction of eosinolphil, the release ofimmunoglobulin and inflammatory chemokines in plasma and bronchoalveolarfluid as well as the suppression of airway hyperresponsiveness andgolblet cell hyperplasia in a OVA-sensitized/challenged mouse model.

Accordingly, in accordance with the other aspect of the presentinvention, present invention provide a pharmaceutical compositioncomprising the purified extract containing abundant active ingredientsprepared by the above-described methods from Pseudolysimachion rotundumvar subintegrum as an active ingredient for the treatment and preventionof inflammatory, allergic or asthmatic disease.

Present invention provide a pharmaceutical composition comprising thepurified extract containing abundant active ingredients prepared by theabove-described methods from Pseudolysimachion rotundum var subintegrumas an active ingredient and the pharmaceutically acceptable carriers orexcipients, for the treatment and prevention of inflammatory, allergicor asthmatic disease.

In accordance with another aspect of the present invention, there isalso provided a use of the purified extract containing abundant activeingredients prepared by the above-described methods fromPseudolysimachion rotundum var subintegrum for manufacture of medicinesemployed for treating or preventing inflammatory, allergic or asthmaticdisease.

The term “pharmaceutically acceptable carriers or excipients” definedherein comprises “pharmaceutical additives, the inactive ingredientsused to make up a medication. They include dyes, flavors, binders,emollients, fillers, lubricants, preservatives, and many moreclassifications. Common excipients include cornstarch, lactose, talc,magnesium stearate, sucrose, gelatin, calcium stearate, silicon dioxide,shellac and glaze, which has been well-known in the art (See, Home-pageof Food and Drug Administration: fda.gov or drug information online:drugs.com) or previous literature (for example, Rowe, Raymond C et al.,Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 7thEdition, 2012)

In accordance with another aspect of the present invention, there isalso provided a method of treating or preventing inflammatory, allergicor asthmatic disease in mammals, wherein the method comprisesadministering a therapeutically effective amount of the purified extractcontaining abundant active ingredients prepared by the above-describedmethods from Pseudolysimachion rotundum var subintegrum into the mammalsuffering from inflammatory, allergic or asthmatic diseases.

The inventive composition for treating and preventing inflammatory,allergic or asthmatic disease may comprises above extracts as 0.1˜99%,preferably, 0.1˜50% by weight based on the total weight of thecomposition.

The composition according to the present invention can be provided as apharmaceutical composition containing pharmaceutically acceptablecarriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acaciarubber, alginate, gelatin, calcium phosphate, calcium silicate,cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineraloil. The formulations may additionally include fillers,anti-agglutinating agents, lubricating agents, wetting agents, flavoringagents, emulsifiers, preservatives and the like. The compositions of theinvention may be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after their administration to a patientby employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolvedin oils, propylene glycol or other solvents that are commonly used toproduce an injection. Suitable examples of the carriers includephysiological saline, polyethylene glycol, ethanol, vegetable oils,isopropyl myristate, etc., but are not limited to them. For topicaladministration, the extract of the present invention can be formulatedin the form of ointments and creams.

Pharmaceutical formulations containing present composition may beprepared in any form, such as oral dosage form (powder, tablet, capsule,soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet,granule), or topical preparation (cream, ointment, lotion, gel, balm,patch, paste, spray solution, aerosol and the like), or injectablepreparation (solution, suspension, emulsion).

The composition of the present invention in pharmaceutical dosage formsmay be used in the form of their pharmaceutically acceptable salts, andalso may be used alone or in appropriate association, as well as incombination with other pharmaceutically active compounds.

The desirable dose of the inventive extract varies depending on thecondition and the weight of the subject, severity, drug form, route andperiod of administration, and may be chosen by those skilled in the art.However, in order to obtain desirable effects, it is generallyrecommended to administer at the amount ranging from 0.0001 to 1000mg/kg, preferably, 0.001 to 100 mg/kg by weight/day of the inventiveextract of the present invention. The dose may be administered in singleor divided into several times per day.

The pharmaceutical composition of present invention can be administeredto a subject animal such as mammals (rat, mouse, domestic animals orhuman) via various routes. All modes of administration are contemplated,for example, administration can be made orally, rectally or byintravenous, intramuscular, subcutaneous, intracutaneous, intrathecal,epidural or intracerebroventricular injection.

The inventive extract of the present invention also can be used as amain component or additive and aiding agent in the preparation ofvarious functional health food and health care food.

Accordingly, it is the other object of the present invention to providea health functional food comprising the purified extract containingabundant active ingredients prepared by the above-described methods fromPseudolysimachion rotundum var subintegrum for the prevention oralleviation of inflammatory, allergic or asthmatic disease.

The term “a functional health food” defined herein” the functional foodhaving enhanced functionality such as physical functionality orphysiological functionality by adding the extract of the presentinvention to conventional food to prevent or improve the purposeddiseases in human or mammal.

It is the other object of the present invention to provide a health carefood comprising the purified extract containing abundant activeingredients prepared by the above-described methods fromPseudolysimachion rotundum var subintegrum, together with asitologically acceptable additive for the prevention or alleviation ofinflammatory, allergic or asthmatic disease.

The term “a health care food” defined herein “the food containing theextract of the present invention showing no specific intended effect butgeneral intended effect in a small amount of quantity as a form ofadditive or in a whole amount of quantity as a form of powder, granule,capsule, pill, tablet etc.

The term “a sitologically acceptable additive” defined herein comprises“any substance the intended use which results or may reasonably beexpected to result-directly or indirectly-in its becoming a component orotherwise affecting the characteristics of any food”, and can beclassified into three groups according to its origin, i.e., (1)chemically synthetic additive such as ketones, glycine, potassiumcitrate, nicotinic acid, etc; (2) natural additive such as persimmondye, licorice extract, crystalline cellulose, gua dum etc; (3) the mixedadditive therewith such as sodium L-glutamate, preservatives, tar dyeetc, or various categories according to its function in the food, forexample, thickening agent, maturing agent, bleaching agent, sequestrant,humectant, anti-caking agent, clarifying agents, curing agent,emulsifier, stabilizer, thickener, bases and acid, foaming agents,nutrients, coloring agent, flavoring agent, sweetener, preservativeagent, anti-oxidant, etc, which has been well-known in the art orprevious literature (See, “Codex General Standard for Food Additives”(GSFA, Codex STAN 192-1995) in Home-page of GSFA Online:codexalimentarius.net/gsfaonline).

If a substance is added to a food for a specific purpose in that food,it is referred to as a direct additive and indirect food additives arethose that become part of the food in trace amounts due to itspackaging, storage or other handling.

The term “health care foods or health functional foods” disclosed hereincan be contained in food, health beverage, dietary supplement etc, andmay be formulated into a form of pharmaceutically dosing form such as apowder, granule, tablet, suspension, emulsion, syrup, chewing tablet,capsule, beverage etc; or the food form, for example, bread, rice cake,dry fruit, candy, chocolate, chewing gum, ice cream, milk such aslow-fat milk, lactose-hydrolyzed milk, goat-milk, processed milk, milkproduct such as fermented milk, butter, concentrated milk, milk cream,butter oil, natural cheese, processed cheese, dry milk, milk serum etc,processed meat product such as hamburger, ham, sausage, bacon etc,processed egg product, fish meat product such as fish cake etc, noodleproducts such as instant noodles, dried noodles, wet noodles, friednoodles, non-fried noodles, gelatinized dry noodles, cooked noodles,frozen noodles, Pasta etc, tea product such as tea bag, leached tea etc,health drinks such as fruit drinks, vegetable drinks, carbonated softdrinks, soymilk drinks, lactic beverage mixed beverage, etc, seasoningfood such as soy sauce, soybean paste, red pepper paste, chunjang (akind of fermented soybean product colored by caramel), cheonggukjang(natural fermented soybean by B. subtillis), mixed paste, vinegar,sauce, ketchup, curry, dressing etc, margarine, shortening, pizza etc,but not intended herein to limit thereto, for preventing or improving ofpurposed disease.

Also, above described extract can be added to food or beverage forprevention and improvement of purposed disorder. The amount of abovedescribed extract in food or beverage as a functional health food orhealth care food may generally range from about 0.01 to 100 w/w % oftotal weight of food for functional health food composition. Inparticular, although the preferable amount of the extract of the presentinvention in the functional health food, health care food or specialnutrient food may be varied in accordance to the intended purpose ofeach food, it is preferably used in general to use as an additive in theamount of the extract of the present invention ranging from about 0.01to 5% in food such as noodles and the like, from 40 to 100% in healthcare food on the ratio of 100% of the food composition.

Providing that the health beverage composition of present inventioncontains above described extract or compound as an essential componentin the indicated ratio, there is no particular limitation on the otherliquid component, wherein the other component can be various deodorantor natural carbohydrate etc such as conventional beverage. Examples ofaforementioned natural carbohydrate are monosaccharide such as glucose,fructose etc; disaccharide such as maltose, sucrose etc; conventionalsugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol,and erythritol etc. As the other deodorant than aforementioned ones,natural deodorant such as taumatin, stevia extract such as levaudiosideA, glycyrrhizin et al., and synthetic deodorant such as saccharin,aspartame et al., may be useful favorably. The amount of above describednatural carbohydrate is generally ranges from about 1 to 20 g,preferably 5 to 12 g in the ratio of 100 Ml of present beveragecomposition.

The other components than aforementioned composition are variousnutrients, a vitamin, a mineral or an electrolyte, synthetic flavoringagent, a coloring agent and improving agent in case of cheese, chocolateet al., pectic acid and the salt thereof, alginic acid and the saltthereof, organic acid, protective colloidal adhesive, pH controllingagent, stabilizer, a preservative, glycerin, alcohol, carbonizing agentused in carbonate beverage et al. The other component thanaforementioned ones may be fruit juice for preparing natural fruitjuice, fruit juice beverage and vegetable beverage, wherein thecomponent can be used independently or in combination. The ratio of thecomponents is not so important but is generally range from about 0 to 20w/w % per 100 w/w % present composition. Examples of addable foodcomprising aforementioned extract or compound therein are various food,beverage, gum, vitamin complex, health improving food and the like.

Inventive extract of the present invention has no toxicity and adverseeffect therefore; they can be used with safe.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

The present invention is more specifically explained by the followingexamples. However, it should be understood that the present invention isnot limited to these examples in any manner.

Advantageous Effects

As described in the present invention, inventive novel industrializedmethod for preparing purified extract containing more abundant activeingredients such as catalpol derivatives from the extract ofPseudolysimachion rotundum var subintegrum and the purified extractshowed more potent anti-inflammatory, anti-allergy and anti-asthmaactivity than that prepared by the conventional preparation methoddisclosed in the prior art through various in vivo tests such asinhibition test on the reproduction of eosinolphil, the release ofimmunoglobulin and inflammatory chemokines in plasma and bronchoalveolarfluid as well as the suppression of airway hyperresponsiveness andgolblet cell hyperplasia in a OVA-sensitized/challenged mouse model.Therefore, it can be used as the therapeutics or functional health foodfor treating and preventing inflammatory, allergic or asthmatic disease.

DESCRIPTION OF DRAWINGS

The above and other objects, features and other advantages of thepresent invention will more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which;

FIG. 1 shows HPLC analysis of the crude extract of Pseudolysimachionrotundum var subintegrum prepared in comparative Example 1;

FIG. 2 shows HPLC analysis of the inventive purified extract (ATC1) ofPseudolysimachion rotundum var subintegrum prepared in Example 1;

FIG. 3 shows HPLC analysis of the inventive purified extract (ATC2) ofPseudolysimachion rotundum var subintegrum prepared in Example 2;

FIG. 4 shows the suppressive effect of the inventive purified extract onairway hyperresponsiveness in a OVA-sensitized/challenged mouse model;

FIG. 5 shows the inhibitory effect of the inventive purified extract onthe recruitment of inflammatory cells in bronchoalveolar lavage fluid;

FIG. 6 shows the inhibitory effect of the inventive purified extract onthe release of immunoglobulin (total IgE) in blood serum;

FIG. 7 shows the inhibitory effect of the inventive purified extract onthe release of OVA-specific IgE in blood serum;

FIG. 8 represents the inhibitory effect of the inventive purifiedextract on the release of IL-1 beta;

FIG. 9 presents the inhibitory effect of the inventive purified extracton the release of inflammatory chemokines;

FIG. 10 represents the inhibitory effect of the inventive purifiedextract on the recruitment of inflammatory cells on lung tissue cellusing by the histological examination of bronchoalveolar lavage;

FIG. 11 represents the inhibitory effect of the inventive purifiedextract on the mucus secretion in lung tissue cell using by thehistological examination of bronchoalveolar lavage;

FIGS. 12 and 13 represent the inhibitory effect of the inventivepurified extract on the recruitment of inflammatory cells on lung tissuecell using by the histological examination of bronchoalveolar lavage;

FIGS. 14 and 15 represent the inhibitory effect of the inventivepurified extract on the mucus secretion in lung tissue cell using by thehistological examination of bronchoalveolar lavage.

BEST MODE FOR CARRYING OUT THE INVENTION

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

The present invention is more specifically explained by the followingexamples. However, it should be understood that the present invention isnot limited to these examples in any manner.

EXAMPLES

The following Reference Example, Examples and Experimental Examples areintended to further illustrate the present invention without limitingits scope.

Comparative Example 1. Preparation of the Crude Extract ofPseudolysimachion rotundum Var subintegrum

1-1. Preparation of Crude Extract (ATE)

1 kg of dried Pseudolysimachion rotundum var subintegrum (cultivated at244, Soi-myeon Eumseong-gun Chungcheongbuk-do in Korea according to GAP)cut into small pieces and mixed with 10 L of 40% ethanol. The mixturewas stirred at room temperature for 24 hours and extracted with refluxextraction at 78° C. for 12 hours to collect the filtrate, three times.The extract was filtered with filter paper to remove the debris. Thecollected filtrate was concentrated by rotary evaporator (EYELA, N-2100,Japan) at 55˜65° C. under reduced pressure and dried with freezing dryerto obtain 202 g of dried crude extract (designated as ‘ACE” hereinafter)for used as a comparative example.

1-2. Preparation of Crude Extract (ATM)

1.1 kg of dried Pseudolysimachion rotundum var subintegrum (cultivatedat 244, Soi-myeon Eumseong-gun Chungcheongbuk-do in Korea according toGAP) cut into small pieces and mixed with 5 L of methanol. The mixturewas stirred at room temperature for 24 hours and extracted with refluxextraction at 78° C. for 12 hours to collect the filtrate, three times.The extract was filtered with filter paper to remove the debris. Thecollected filtrate was concentrated by rotary evaporator (EYELA, N-2100,Japan) at 55˜65° C. under reduced pressure and dried with freezing dryerto obtain 100.5 g of dried crude extract (designated as ‘ACM”hereinafter) for used as a comparative example.

1-3. Component Analysis

The component analysis was performed using by HPLC (Agilent 1260 model,USA) according to the condition in Table 1 and the result was shown inFIG. 1.

As can be seem in FIG. 1, it has been confirmed that each ingredient wasdetected at 9.548 mins (Verproside), 10.817 mins (Veratric acid), 16.728mins (Catalposide), 20.346 min (Picroside II), 21.853 mins (Isovanilloylcatalpol), and 30.462 mins (6-O-veratrolyl catalpol) respectively.

The content of each ingredient (%) in the sample was calculated based onthe HPLC pattern (retention time) according to math formulae 1.content of each ingredient=conc. of standard (mg/rip/conc. of testsample (mg/ml)×At/As×purity of standard (%)  Math formulae 1wherein “At” denotes the ingredient area in test sample and “As” denotesthat in standard provided that the sampled volume of test sample andstandard is identical to each other.

TABLE 1 HPLC condition HPLC condition Pump Agilent 1260 Series, 1260quart pump Detector Agilent 1260 Series, 1260 DAD Column Agilent EclipseXOB C18, 4.6 × 50 cm, 5 μm Flow rate 1.5 ml/min UV Absorbance 266 nmMobile phase A: phosphate buffer (pH = 3.5) Mobile phase B: methanolMobile phase A Mobile phase B Time (%) (%) Mobile phase  0~5 80 20  5~2075 25 20~25 75 25 25~30 55 45 30~35 55 45 35~36 80 20 36~40 80 20Injection volume 10 μl

At the result, it has been confirmed that the crude extract ofPseudolysimachion rotundum var subintegrum contains only 8.49% (w/w)catalposide derivatives, i.e., 5.9% (w/w) verproside, 0.21% (w/w)veratric acid, 0.82% (w/w) catalposide, 0.40% (w/w) picroside II, 0.42%(w/w) isovanillyl catalpol, and 0.74% (w/w) 6-O-veratroyl catalpol,respectively, as can be seen in Table 2.

TABLE 2 HPLC result (crude extract: ACE) Comparative Example 1 Activeingredient Retention Time (mins) Content (w/w %) Verproside 9.548 5.90Veratric acid 10.817 0.21 Catalposide 16.728 0.82 Picroside II 20.3460.40 Insovanilloyl catalpol 21.853 0.42 6-O-veratroyl catalpol 30.4620.74 Total 8.49

Example 1. Preparation of the Purified Extract (ATC1) ofPseudolysimachion rotundum Var subintegrum

The crude extract (ACE) of Pseudolysimachion rotundum var subintegrumprepared by the conventional method according to Comparative Example 1,was suspended in 2 L of distilled water and the suspension was addedwith 2 L of butanol to fractionate into butanol-soluble fraction andwater-soluble fraction. The butanol soluble fraction was collected,concentrated under reduced pressure and dried to afford 82 g of theinventive purified extract fractionated with butanol (ATC1) used as atest example.

The component analysis was performed using by HPLC (Agilent 1260 model,USA) according to the condition in Table 1 and the result was shown inFIG. 2.

As can be seem in FIG. 2, it has been confirmed that each ingredient wasdetected at 9.545 mins (Verproside), 10.821 mins (Veratric acid), 16.727mins (Catalposide), 20.345 min (Picroside II), 21.853 mins (Isovanilloylcatalpol), and 30.462 mins (6-O-veratroyl catalpol) respectively.

The content of each ingredient (%) in the sample was calculated based onthe HPLC pattern (retention time) according to math formulae 1.

At the result, it has been confirmed that the inventive purified extractfractionated with butanol (ATC1) of Pseudolysimachion rotundum varsubintegrum contains 25.64% (w/w) catalposide derivatives, i.e., 17.60%(w/w) verproside, 0.72% (w/w) veratric acid, 2.62% (w/w) catalposide,1.08% (w/w) picroside II, 1.26% (w/w) isovanillyl catalpol, and 2.36%(w/w) 6-O-veratroyl catalpol, respectively, as can be seen in Table 3.

TABLE 3 HPLC result (purified extract: ATC1) Example 1 Active ingredientRetention Time (mins) Content (w/w %) Verproside 9.545 17.60 Veratricacid 10.821 0.72 Catalposide 16.727 2.62 Picroside II 20.345 1.08Insovanilloyl catalpol 21.853 1.26 6-O-veratroyl catalpol 30.462 2.36Total 25.64

Example 2. Preparation of the Purified Extract (ATC2) ofPseudolysimachion rotundum Var subintegrum

The inventive purified extract fractionated with butanol (ATC1) ofPseudolysimachion rotundum var subintegrum according to Example 1, wasdissolved in 75 mL of mixed solvent (distilled water:methanol=1:0.003)and 75 g of the solution was loaded on reverse phase columnchromatography (C18(IV)-D-75-120 nm, AGC Si-Tech Co. Ltd., Japan, 450 g)with eluting the suspension using by eluting solvent (distilledwater:methanol=90:10→60:40). 8.4 L of the eluted solution running at theinitial eluting solvent system (distilled water:methanol=90:10) wascollected and concentrated under reduced pressure. 5.6 L of the elutedsolution running at the late eluting solvent system (distilledwater:methanol=60:40) was collected, concentrated under reduced pressureand dried to afford 33 g of the inventive purified extract with thesecondary fractionation (ATC2) used as a test example.

The component analysis was performed using by HPLC (Agilent 1260 model,USA) according to the condition in Table 1 and the result was shown inFIG. 3.

As can be seem in FIG. 3, it has been confirmed that each ingredient wasdetected at 9.525 mins (Verproside), 10.818 mins (Veratric acid), 16.721mins (Catalposide), 20.346 min (Picroside II), 21.857 mins (Isovanilloylcatalpol), and 30.462 mins (6-O-veratroyl catalpol) respectively.

The content of each ingredient (%) in the sample was calculated based onthe HPLC pattern (retention time) according to math formulae 1.

At the result, it has been confirmed that the inventive purified extractwith the secondary fractionation (ATC2) of Pseudolysimachion rotundumvar subintegrum contains 65.63% (w/w) catalposide derivatives, i.e.,43.83% (w/w) verproside, 1.80% (w/w) veratric acid, 7.07% (w/w)catalposide, 2.93% (w/w) picroside II, 3.85% (w/w) isovanillyl catalpol,and 6.15% (w/w) 6-O-veratroyl catalpol, respectively, as can be seen inTable 4.

TABLE 4 HPLC result (purified extract: ATC2) Example 2 Active ingredientRetention Time (mins) Content (w/w %) Verproside 9.524 43.83 Veratricacid 10.818 1.80 Catalposide 16.721 7.07 Picroside II 20.346 2.93Insovanilloyl catalpol 21.857 3.85 6-O-veratroyl catalpol 30.462 6.15Total 65.63

Experimental Example 1. Preliminary Determination of the Total Serum IgELevel in OVA-Sensitized/Challenged Mouse Model

In order to found the purified extract showing more pharmacologicallypotent activity than the crude extract prepared in comparative Example,following preliminary test was performed by the method disclosed in theliterature (Elias, J. A. et al., J. Clin. Invest., 111, pp 297-297,2003).

1-1. Animal Sensitization and Airway Challenge

Specific pathogen-free female BALB/c mice (about 20 g), aged 6 weeks,which were routinely screened serologically for relevant respiratorypathogens, were purchased from ORIENT Co. (Seoul, Korea) and acclimatedwith the experimental environment for 1 week.

Briefly, mice were sensitized by intraperitoneal injection of 20 μg OVA(Ovalbumin; A5503, Sigma, St. Louis, Mo.), which was emulsified in 2 mgaluminum hydroxide in 200 μl of PBS buffer (pH 7.4), biweekly. The micewere challenged through the airways with OVA (1% in PBS) for 30 minusing an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan) fromthe 28th day to 34th day after the initial sensitization. 24 hrs afterthe antigen treatment, the airway hyperresponsiveness was determined andthe mice were sacrificed 48 hrs after the last challenge. The mice weresacrificed with an overdose of pentobarbital (Entobal®, Hanrim Pharm.Co. Ltd.) 24 h after the last challenge, and a tracheotomy wasperformed. After 1.2 ml of physiological saline solution (PBS) wasinstilled into the lungs, bronchoalveolar lavage fluid (BALF) wasobtained by aspiration three times (total 1.5 ml) via trachealcannulation.

The groups were divided into several groups, i.e., (a) normal controlgroup (NC): the groups treated or not-treated with OVA; (b)asthma-induced group(OVA): the groups treated with OVA to induce asthma;and (c) comparative group: the groups treated with positive controlgroup (M30, montelukast; 30 mg/kg, PO, Sigma-Aldrich Co. Ltd., SML 0101)1 hr prior to OVA inhalation.

The test group consists of 6 mice for each group and 1 hour prior to OVAinhalation, various concentrations of the test sample, ATC1 (30 mg/kgand 100 mg/kg) and ATM (30 and 100 mg/kg) were orally administrated tothe mice.

As shown in Table 5, the total level of IgE in blood serum inasthma-induced group (OVA) was significantly increased whereas those inthe test sample group orally administrated with various concentrationsof test samples (ATC1, 30 mg/kg and 100 mg/kg) were more reduced thanthat in the group treated with crude extract of Pseudolysimachionrotundum var subintegrum (ATM, 30 and 100 mg/kg). (See Table 5)

TABLE 5 The level of total IgE in blood serum total IgE (mg/ml) NC 0.57± 0.02 OVA 5.23 ± 0.34 ATC1  30 mg/kg 2.05 ± 0.12 100 mg/kg 2.14 ± 0.25ATM  30 mg/kg 3.09 ± 0.54 100 mg/kg 2.49 ± 0.35 Monte30 1.82 ± 0.40

Experimental Example 2. Anti-Asthamtic Effect Using by AirwayHyperresponsiveness Test in a OVA-Sensitized/Challenged Mouse Model

In order to confirm the anti-asthmatic effect of test samples preparedin Examples using by airway hyperresponsiveness test in aOVA-sensitized/challenged mouse model, following test was performed bythe method disclosed in the literature (Elias, J. A. et al., J. Clin.Invest., 111, pp 297-297, 2003).

1-1. Animal Sensitization and Airway Challenge

Specific pathogen-free female BALB/c mice (about 20 g), aged 6 weeks,which were routinely screened serologically for relevant respiratorypathogens, were purchased from ORIENT Co. (Seoul, Korea) and acclimatedwith the experimental environment for 1 week.

Briefly, mice were sensitized by intraperitoneal injection of 20 μg OVA(Ovalbumin; A5503, Sigma, St. Louis, Mo.), which was emulsified in 2 mgaluminum hydroxide in 200 μl of PBS buffer (pH 7.4), biweekly. The micewere challenged through the airways with OVA (1% in PBS) for 30 minusing an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan) fromthe 28th day to 34th day after the initial sensitization. 24 hrs afterthe antigen treatment, the airway hyperresponsiveness was determined andthe mice were sacrificed 48 hrs after the last challenge. The mice weresacrificed with an overdose of pentobarbital (Entobal®, Hanrim Pharm.Co. Ltd.) 24 h after the last challenge, and a tracheotomy wasperformed. After 1.2 ml of physiological saline solution (PBS) wasinstilled into the lungs, bronchoalveolar lavage fluid (BALF) wasobtained by aspiration three times (total 1.5 ml) via trachealcannulation.

Group of mice (n=6) were studied; they received the following treatment:(1) The non-treatment group with OVA as a normal control group (NC); (2)The control group treated and inhaled with OVA as an asthma inducedgroup (OVA); (3) The positive control group treated with known asthmatherapeutics (Montelukast; 30 mg/kg, PO, Sigma-Aldrich Korea, SML-0101,M30) 1 hour prior to OVA inhalation; (4) The test sample group orallyadministrated with various concentrations of test samples, i.e., 5mg/kg, 10 mg/kg, 25 mg/kg and 50 mg/kg of purified extract (ATC2) 1 hourprior to OVA inhalation.

1-2. Evaluation of Airway Hyperresponsiveness

In order to evaluate the airway hyperresponsiveness of the mice, theairway resistance was determined using by apparatus (One chamber wholebody plethymography, OCP3000, All Medicus, Seoul. Korea) and thedetermined value was statistically calculated by Pehn value (EnhancePause) reflecting on the degree of airway obstruction. The Penh valuewas determined for 3 mins by the process of determining the basal valueat the eupnea phase and determining the Penh value after inhaling PBSwith Ultrasonic nebulizer (NE-U12, IMRON Corp., Tokyo, JAPAN) for 3mins.

Thereafter, various concentrations of methacholine (A2251, Sigma, St.Louis, Mo.), 12, 25 and 50 mg/ml, were inhaled with increasingconcentration to determine the Pehn values. The increase of Penh valuewas expressed as percentage (%) after the methacholine inhalation andthe Penh value of basal line was set to 100%. The value of Pehn wascalculated according to math formulae 2 and the result was shown in FIG.4.Pehn=(Te/RT−1)×PEF/PIF  Math formulae 2

Te: Expiration Time (The period from a inhalation to the nextinhalation);

RT: Relaxation Time (The period that the exhaled volume is reached tothe extent to 30% of one expiration volume during expiration)

PEF: Peak Expiration Flow

PIF: Peak Inspiration Flow

At the result, it has been confirmed that the Penh value in the controlgroup treated and inhaled with OVA as an asthma induced group (OVA) wassharply increased while that in the non-treatment group with OVA as anormal control group (NC) had been gradually increased with increasingthe concentration of methancholine.

In a while, the Penh value in the positive control group treated withMontelukast (MO) as well as the test sample group orally administratedwith various concentrations of test samples (ATC-10, ATC-25, and ATC-50)were significantly reduced regardless of the concentration ofmethacholine. (See Table 6)

TABLE 6 Penh Value Methacholine (Conc. mg/Ml) 0 12.5 25 50 NC 0.37 ±0.03 0.39 ± 0.04 0.69 ± 0.14 1.07 ± 0.18 OVA 0.85 ± 0.10 2.79 ± 0.255.99 ± 0.92 7.24 ± 0.74 ATC2  5 0.52 ± 0.05 1.69 ± 0.27 2.68 ± 0.52 5.19± 0.74 (mg/kg) 10 0.63 ± 0.06 1.54 ± 0.18 2.01 ± 0.52 3.08 ± 0.40 250.64 ± 0.08 1.31 ± 0.17 1.64 ± 0.31 2.43 ± 0.33 50 0.51 ± 0.06 1.02 ±0.17   1.36 ± 0.09/ 1.82 ± 0.28 M30 0.49 ± 0.04 1.54 ± 0.20 1.98 ± 0.372.51 ± 0.36

It has been confirmed that those change in Penh value has been prominentin case of higher-dose methacholine treatment group rather than inlower-dose methacholine treatment group and the Penh value in the testsample for the same concentration of methacholine, has been remarkablydecreased in a dose dependent manner.

Accordingly, it has been confirmed that the inventive purified extracteffectively suppressed the airway hyperresponsiveness and therefore,they are useful in treating or preventing the asthma disease, anallergic disease in airway.

Experimental Example 3. Effect on the Level of Eosinophil andInflammatory Cells in BALF

In order to confirm the inhibition effect of test samples prepared inExamples on the level of eosinophil and inflammatory cells inbronchoalveolar fluid (BALF), following test was performed by the methoddisclosed in the literature (Chen M. et al., Immunolgy, pp 376-384,2011).

The bronchoalveolar lavage fluid (BALF) prepared in Experimental Example1 was recovered to determine the level of inflammatory cells.

The total inflammatory cell number was assessed by the counting of cellsin at least five squares of a hemocytometer after excluding dead cellsby staining with trypan blue (Daigle I. et al., Swiss Med Wkly, 131, pp231-7, 2001). 100 μl of BALF was loaded onto a slide and centrifuged(200×g, 4° C., 10 min) to fix the cells onto the slide using a Cellspinmachine (Cyto12.5+clip5, Hanil Science Industrial, Korea). The cellswere stained by Diff-Quick® Stain reagents (Sysmex, Cat No. 38721,Switzerland) according to the manufacturer's instructions. Statisticalsignificance was determined by Student's two-tailed t-test forindependent means and the critical level for significance was set atP<0.05.

As shown in FIG. 5, the total number of eosinophil and inflammatorycells in the control group treated and inhaled with OVA as an asthmainduced group (OVA) was significantly increased comparing with those inthe non-treatment group with OVA as a normal control group (NC).

The total number of eosinophil and inflammatory cells in the positivecontrol group treated with Montelukast (MO) as well as the test samplegroup orally administrated with various concentrations of test samples(ATC-5, ATC-10, ATC-25, and ATC-50) were significantly reduced. (SeeTable 7)

TABLE 7 total number of eosinophil and inflammatory cells in BALF No. ofinflammatory cells No. of inflammatory (10³ cells/mouse) No. ofeosinolphil cells NC 0.00 ± 0.00 8.28 ± 1.46 OVA 135.44 ± 4.54  260.48 ±10.39  ATC2  5 72.23 ± 9.45  158.2 ± 15.38 (mg/kg) 10 55.40 ± 3.46 131.67 ± 9.03  25 40.8 ± 2.34 98.6 ± 4.57 50 36.57 ± 4.02  88.1 ± 7.04M30 52.03 ± 4.06  106.67 ± 6.48 

Experimental Example 4. Effect on the Level of IgE and OVA-Specific IgEin Blood Serum

In order to confirm the inhibition effect of test samples prepared inExamples on the level of IgE and OVA-specific IgE in blood serum,following test was performed by the method disclosed in the literature(Kay, A. B., The New England Journal of Medicine, 344, pp 30-37, 2001).

The blood serum and bronchoalveolar lavage fluid (BALF) prepared inExperimental Example 1 was recovered to determine the level of IgE andOVA-specific IgE in blood serum.

The blood serum and bronchoalveolar lavage fluid (BALF) was added to96-well plates (ELISA plate) and coated with 0.1M NaHCO₃ buffer solution(pH 8.3) containing 20 μg/mlof OVA (Sigma, Mo., USA) at 4° C. overnight.After inhibiting nonspecific reaction using by PBS containing 1% bovineserum albumin, the serum for testing was diluted to 1:400 and reactedtogether for 2 hours at room temperature. After washing, the serum wasreacted with diluted (×300) anti-mouse IgE monoclonal antibody (MCA419,Serotec, Oxford, UK) for 2 hours and with diluted (×4000) HRP-conjugatedgoat anti-rat IgG polyclonal A (STAR110P, Serotec, UK) for 1 hours atroom temperature. After washing, the solution was stained with3,3′,5,5′-tetramethylbenzidine (52-00-02, KPL) substrate and thereaction was stopped by 2N H₂SO₄ to determine the absorbance using byspectroscopy (Versamax, Molecular Devices, US) at 450 nm.

As shown in FIG. 6 and FIG. 7, the level of IgE and OVA-specific IgE inblood serum in the control group treated and inhaled with OVA as anasthma induced group (OVA) was significantly increased whereas those inthe positive control group treated with Montelukast (MO) as well as thetest sample group orally administrated with various concentrations oftest samples (ATC-5, ATC-10, ATC-25, and ATC-50) were significantlyreduced. (See Table 8)

TABLE 8 The level of IgE and OVA-specific IgE in blood serumConcentration level of OVA-specific (μg/ml) level of IgE in serum IgE NC0.92 ± 0.17 0.05 ± 0.00 OVA 7.68 ± 0.42 0.17 ± 0.02 ATC2  5 6.51 ± 0.720.13 ± 0.01 (mg/kg) 10 4.97 ± 0.91 0.10 ± 0.01 25 4.56 ± 0.73 0.09 ±0.02 50 4.01 ± 0.67 0.08 ± 0.01 M30 4.76 ± 0.73 0.09 ± 0.02

Accordingly, it has been confirmed that the inventive purified extracteffectively inhibited the level of IgE and OVA-specific IgE in bloodserum and therefore, they are useful in treating or preventing theallergic disease and asthma disease.

Experimental Example 5. Effect on the Level of Inflammatory Cytokines inBALF

In order to confirm the inhibition effect of test samples prepared inExamples on the level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1βin bronchoalveolar lavage fluid (BALF), following test was performed bythe sandwich enzyme immunosorbent assay method disclosed in theliterature (Renz H. et al., J. Exp. Med., 1777, pp 1175-1180, 1993).

The blood serum and bronchoalveolar lavage fluid (BALF) was added to96-well plates (ELISA plate) coated with cytokine antibody to induceantigen-antibody reaction for 2 hours at room temperature. The level ofTh2 cytokines (IL-4, IL-5 and IL-13) and IL-1β in bronchoalveolar lavagefluid (BALF) was determined using by ELISA kit (Biosource Int. CA, USA)specifically reacting with each cytokine according to the manufacture'smanual.

As shown in FIG. 8 and FIG. 9, 48 hours after the OVA-treatment, thelevel of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1β in the controlgroup treated and inhaled with OVA as an asthma induced group (OVA) wassignificantly increased comparing with those in the non-treatment groupwith OVA as a normal control group (NC).

The increased level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1β inthe positive control group treated with Montelukast (MO, 30 mg/kg) aswell as the test sample group orally administrated with variousconcentrations of test samples (ATC-10, ATC-25, and ATC-50) weresignificantly reduced. (See Table 9)

TABLE 9 The level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1βconc. (pg/ml) IL-1β IL-4 IL-5 IL-13 NC  94.12 ± 65.24 20.94 ± 1.76 57.51± 3.15  22.25 ± 3.04 OVA 281.78 ± 26.15 48.76 ± 6.96 109.48 ± 2.87 44.76 ± 4.85 ATC2  5 243.93 ± 27.58 35.27 ± 5.26 90.77 ± 12.78 34.29 ±7.55 (mg/kg) 10 226.33 ± 14.21 35.18 ± 4.45 79.26 ± 9.60  29.50 ± 2.7625 190.30 ± 17.82 29.94 ± 2.32 73.74 ± 9.54  27.27 ± 4.42 50 170.70 ±25.43 25.26 ± 5.55 57.92 ± 19.99 23.53 ± 4.10 M30 187.03 ± 47.17 32.60 ±4.53 69.74 ± 7.17  25.93 ± 4.13

Accordingly, it has been confirmed that the inventive purified extracteffectively inhibited the level of level of Th2 cytokines (IL-4, IL-5and IL-13) and IL-1β in BALF and therefore, they are useful in treatingor preventing the allergic disease and asthma disease.

Experimental Example 6. Lung Histology

In order to confirm the anti-asthmatic effect of test samples preparedin Examples, following histopathological analysis on broncho-alveolartissue was performed by the method disclosed in the literature (Kwak YG. et al., J. Clin. Invest., 111, pp 1083-1092, 2003).

The delivered lung tissues of BALB/c mice which had not performbroncho-alveolar lavage was fixed for 24 h in 10% neutral-bufferedformalin. After being embedded in paraffin, then made into 4-μmthickness sections, the tissue was stained with H&E solution(hematoxylin; Sigma MHS-16 and eosin, Sigma HT110-I-32) and theinflammation score of five regions in each section chosen in arandomized manner, was determined. (Inflammation score 0: inflamed cellsis not found in bronchial surrounding, Inflammation score 1: inflamedcells is sporadically found in bronchial surrounding, Inflammation score2: thin inflamed cell layer is found in most of bronchial surrounding,Inflammation score 3: thick inflamed cell layer is found in most ofbronchial surrounding).

As shown in FIG. 10, many inflammatory cells including eosinophills werefound in bronchiolar surroundings and hyperplasia of epithelial cells aswell as hypertrophy of tracheal muscle were also found in the controlgroup treated and inhaled with OVA as an asthma induced group (OVA)whereas the invasion of the inflamed cells was significantly reduced inthe positive control group treated with Montelukast (MO, 30 mg/kg) aswell as the test sample group orally administrated with variousconcentrations of test samples (ATC-10, ATC-25, and ATC-50). (See Table10)

TABLE 10 Inflammation score and the ratio of goblet cell in bronchiolarepithelial cell histopathological Inflammation PAS + cells/bronchioleanalysis score (%) NC 0.06 ± 0.05  2.19 ± 0.54 OVA 2.11 ± 0.07 52.75 ±1.42 ATC2  5 1.75 ± 0.17 48.07 ± 1.15 (mg/kg) 10 1.33 ± 0.14 44.59 ±1.60 25 1.17 ± 0.11 38.61 ± 1.74 50 1.08 ± 0.18 35.71 ± 1.14 M30 1.25 ±0.13 39.21 ± 2.34

Experimental Example 7. Evaluation of Goblet Cell Plasia

In order to confirm the anti-asthmatic effect of test samples preparedin Examples, following goblet cell plasia analysis on broncho-alveolartissue was performed by the method disclosed in the literature (Lee K S.et al., FASEB J., 20, pp 455-465, 2006).

The delivered lung tissues of BALB/c mice which had not performbroncho-alveolar lavage was fixed for 24 h in 10% neutral-bufferedformalin. After being embedded in paraffin, then made into 4-μmthickness sections, the tissue was stained with Periodic acid Schiff(PAS stain kit, T-K7308, IMEB, CA, USA) to determine the ratio of gobletcell in bronchiolar epithelial cell

As shown in FIG. 11, the ratio of goblet cell in bronchiolar epithelialcell was significantly increased in the control group treated andinhaled with OVA as an asthma induced group (OVA) comparing with normalcontrol group (Nc) whereas the ratio of goblet cell in bronchiolarepithelial cell was significantly reduced in the positive control grouptreated with Montelukast (MO, 30 mg/kg) as well as the test sample grouporally administrated with various concentrations of test samples(ATC-10, ATC-25, and ATC-50). (See Table 9)

Experimental Example 8. Acute Toxicity Test of Oral Administration inRat

The acute toxicity test was performed by administrating inventiveextract to 6-weeks aged SPF Sprague-Dawley rats.

250 mg/kg, 500 mg/kg, 1000 mg/kg, 5000 mg/kg of inventive extract wasorally administrated to each group consisting of 2 rats and the symptomsof rats were observed for 14 days. After administrating the extract orcompounds, all the clinical changes i.e., mortality, clinical signs,body weight changes was observed and blood test such as haematologicaltest and hematological biochemistry test was performed. The abnormalchanges of abdominal organ and thoracic organ were observed afterautopsy.

There did not show any changes in mortality, clinical signs, body weightchanges and gross findings in any group or either gender. Furthermore,there showed any toxicity in test group treated with 5000 mg/kg ofinventive extract or compounds.

Accordingly, it has been confirmed that the inventive extract preparedin the present invention was potent and safe substance showing LD₅₀(more than 5000 mg/kg) in oral administration.

MODE FOR INVENTION

Hereinafter, the formulating methods and kinds of excipients will bedescribed, but the present invention is not limited to them. Therepresentative preparation examples were described as follows.

Preparation of Injection

ATC1 extract 100 mg

Sodium metabisulfite 3.0 mg

Methyl paraben 0.8 mg

Propyl paraben 0.1 mg

Distilled water for injection optimum amount

Injection preparation was prepared by dissolving active component,controlling pH to about 7.5 and then filling all the components in 2 Mlample and sterilizing by conventional injection preparation method.

Preparation of Powder

ATC2 extract 500 mg

Corn Starch 100 mg

Lactose 100 mg

Talc 10 mg

Powder preparation was prepared by mixing above components and fillingsealed package.

Preparation of Tablet

ATC1 extract 200 mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components andentabletting.

Preparation of Capsule

ATC2 extract 100 mg

Lactose 50 mg

Corn starch 50 mg

Talc 2 mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components and fillinggelatin capsule by conventional gelatin preparation method.

Preparation of Liquid

ATC1 extract 1000 mg

Sugar 20 g

Polysaccharide 20 g

Lemon flavor 20 g

Liquid preparation was prepared by dissolving active component, and thenfilling all the components in 1000 Ml ample and sterilizing byconventional liquid preparation method.

Preparation of Health Food

ATC2 extract 1000 mg

Vitamin mixture optimum amount

Vitamin A acetate 70 g

Vitamin E 1.0 mg

Vitamin B₁₀. 13 mg

Vitamin B₂ 0.15 mg

Vitamin B6 0.5 mg

Vitamin B1 20.2 g

Vitamin C 10 mg

Biotin 10 g

Amide nicotinic acid 1.7 mg

Folic acid 50 g

Calcium pantothenic acid 0.5 mg

Mineral mixture optimum amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monopotassium phosphate 15 mg

Dicalcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The above mentioned vitamin and mineral mixture may be varied in manyways. Such variations are not to be regarded as a departure from thespirit and scope of the present invention.

Preparation of Health Beverage

ATC1 extract 1000 mg

Citric acid 1000 mg

Oligosaccharide 100 g

Apricot concentration 2 g

Taurine 1 g

Distilled water 900 Ml

Health beverage preparation was prepared by dissolving active component,mixing, stirred at 85° C. for 1 hour, filtered and then filling all thecomponents in 1000 Ml ample and sterilizing by conventional healthbeverage preparation method.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

As described in the present invention, the present invention providesinventive novel industrialized method for preparing purified extractcontaining more abundant active ingredients such as catalpol derivativesfrom the extract of Pseudolysimachion rotundum var subintegrum and thepurified extract showed more potent anti-inflammatory, anti-allergy andanti-asthma activity than that prepared by the conventional preparationmethod disclosed in the prior art through various in vivo tests such asinhibition test on the reproduction of eosinophil, the release ofimmunoglobulin and inflammatory chemokines in plasma and bronchoalveolarfluid as well as the suppression of airway hyperresponsiveness andgoblet cell hyperplasia in a OVA-sensitized/challenged mouse model.Therefore, it can be used as the therapeutics or functional health foodfor treating and preventing inflammatory, allergic or asthmatic disease.

The invention claimed is:
 1. A method of treatment comprising theadministering of a pharmaceutical composition comprising a purifiedextract with the secondary fractionation (ATC2) from the extract ofPseudolysimachion rotundum var subintegrum, comprising 30%-60% (w/w)verproside, 0.5%-10% (w/w) veratric acid, 2%-20% (w/w) catalposide,1%-10% (w/w) picroside II, 1%-10% (w/w) isovanilloyl catalpol and 2%-20%(w/w) 6-O-veratroyl catalpol based on the weight of total extract (100%)of Pseudolysimachion rotundum var subintegrum to a subject in need oftreatment of an ailment selected from the group consisting of aninflammatory disease, an allergy disease and an asthma disease.
 2. Themethod of claim 1, wherein the inflammatory disease is selected from thegroup consisting of eczema, atopic dermatitis, conjunctivitis,periodontal disease, rhinitis, otitis media, laryngopharyngitis,tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease,colitis, hemorrhoid, gout, ankylosing spondylitis, rheumatic fever,systemic lupus erythematosus, fibromyalgia, psoriatic arthritis,osteoarthritis, rheumatic arthritis, periarthritis of shoulder,tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis,cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, chronicinflammatory disease, and acute inflammatory disease; wherein theallergy disease is selected from the group consisting of allergicrhinitis, allergic dermatitis, contact dermatitis, hives, insectallergy, food allergy, drug allergy, allergic conjunctivitis, andhypersensitivity; and wherein the asthma disease is selected from thegroup consisting of sensitivity to dust mites, fur, dandruff, cockroach,food, drug, cough, cigarette smoke, air pollution, food additive,physical activity, exercise, weather change, yellow sand and stress. 3.A method of treatment comprising the administering of a pharmaceuticalcomposition comprising a purified extract with the secondaryfractionation (ATC2) from the extract of Pseudolysimachion rotundum varsubintegrum, comprising 36.5%-91% (w/w) catalpol derivatives selectedfrom the group consisting of verproside, catalposide, picroside II,isovanilloyl catalpol and 6-O-veratroyl catalpol in total extract (100%)of Pseudolysimachion rotundum var subintegrum and which has a relativemixed ratio (w/w) between weight of each catalpol derivative of13.0-16.0 (w/w) verproside, 2.20-2.50 (w/w) catalposide, 1 (w/w)picroside II, 1.10-1.40 (w/w) isovanilloyl catalpol and 2.00-2.20 (w/w)6-O-veratroyl catalpol to a subject in need of treatment of an ailmentselected from the group consisting of an inflammatory disease, anallergy disease and an asthma disease.
 4. The method of claim 3, whereinthe inflammatory disease is selected from the group consisting ofeczema, atopic dermatitis, conjunctivitis, periodontal disease,rhinitis, otitis media, laryngopharyngitis, tonsillitis, pneumonia,gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoid, gout,ankylosing spondylitis, rheumatic fever, systemic lupus erythematosus,fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatic arthritis,periarthritis of shoulder, tendinitis, tenosynovitis, peritendinitis,myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiplesclerosis, chronic inflammatory disease, and acute inflammatory disease;wherein the allergy disease is selected from the group consisting ofallergic rhinitis, allergic dermatitis, contact dermatitis, hives,insect allergy, food allergy, drug allergy, allergic conjunctivitis, andhypersensitivity; and wherein the asthma disease is selected from thegroup consisting of sensitivity to dust mites, fur, dandruff, cockroach,food, drug, cough, cigarette smoke, air pollution, food additive,physical activity, exercise, weather change, yellow sand and stress.